Summary of Work: More than 36 million people are infected by the HIV virus worldwide while 5.3 million new infections occurred during 2000. Although antiviral therapy can extend the life of individuals, the death toll continues to rise: 2.6 million people, the highest number since the epidemic began, died from AIDS this year. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of the mitochondrial DNA polymerase. As many as 40% of patients undergoing AZT treatment develop a mitochondrial dysfunction known as red ragged fiber disease. The mode and effect of the antiviral nucleotide analogs AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication are poorly understood. Which structural properties set this polymerase apart from the nuclear DNA polymerases and give rise to its inhibition patterns is poorly understood. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase gamma (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state Km and kcat values for insertion of 2',3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZT-TP), '?,3'-dideoxy-CTP (ddCTP), 2',3'-didehydro-TTP (D4T-TP), (-)-2',3'-dideoxy-3'-thiacytidine (3TC-TP), and carbocyclic 2',3'-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol gamma in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3'-terminal 3TC residues was 50% as efficient as natural 3'-termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.